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In memory of the 85th birthday of Yakov S. Lebedev (Moscow), who died in 1996, we start this Review on soft-glass matrix effects in donor–acceptor complexes with an appreciation of his pioneering work on high-field EPR spectroscopy on tribochemically generated donor–acceptor complexes. The mechanochemical activation of polycrystalline mixtures of porphyrins (and other donors) and quinone acceptors was found to produce large concentrations of triplet donor molecules and donor–acceptor radical pairs with unusual stability. The Review is continued with reporting on W-band high-field EPR and fast-laser studies on disaccharide matrix effects on structure and dynamics of donor–acceptor protein complexes related to photosynthesis, including the non-oxygenic bacterial reaction center (RC) and the oxygenic RCs Photosystem I (PS I) and Photosystem II (PS II, preliminary results). Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, in particular trehalose and sucrose. Trehalose is most effective in protecting biostructures, both in vivo and in vitro. To clarify the molecular mechanisms of disaccharide bioprotection, structure and dynamics of sucrose and trehalose matrices at different controlled hydration levels were probed by perdeuterated nitroxide spin labels and native cofactor intermediates in their charge-separated states. Trehalose forms a homogeneous amorphous phase in which the hosted molecules are uniformly distributed. Notably, their rotational mobility at room temperature is dramatically impaired by thetrehalose H-bonding network confinement to an extent that in normal protein–matrix systems is only observed at low temperatures around 150K. From the experimental results, formation of an extended H-bonding network of trehalose with protein molecules is inferred, involving both bulk and local water molecules. The H-bond network extends homogeneously over the whole matrix integrating and immobilizing the hosted protein. Taken together, these observations suggest that in photosystems, such as bacterial RCs and PS I complexes, of different size and complexity regarding subunit composition and oligomeric organization, the molecular configuration of the cofactors involved in the primary processes of charge separation is not significantly distorted by incorporation into trehalose glass, even under extensive dehydration. By means of pulsed W-band high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, in conjunction with using isotope labeled water (D2O and H217O), the biologically important issue of sensing and quantification of local water in proteins is addressed. The bacterial RC embedded into the trehalose glass matrix is used as model system. The two native radical cofactor ions of the primary electron donor and acceptor as well as an artificial nitroxide spin label site-specifically attached to the protein surface are studied in the experiments. The three paramagnetic reporter groups probe distinctly different local environments. They sense water molecules via their magnetic hyperfine and quadrupole interactions with either deuterons or 17O nuclei. It is shown that by using oxygen-17 labeled water, quantitative conclusions can be drawn differentiating between local and bulk water. It is concluded that dry trehalose operates as anhydrobiotic protein stabilizer by means of selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Such changes usually have an impact on the global function of a biological system. Finally, preliminary results of optical and W-band EPR experiments on the extremolytes ectoine and its derivative hydroxyectoine are reported; these compounds appear to share several stress-protecting properties with trehalose in terms of stabilizing protein matrices. For instance, they display remarkable stabilizing capabilities towards sensitive proteins and enzymes with respect to freeze-thawing, heat-treatment, and freeze-drying procedures. Moreover, hydroxyectoine is a good glass-forming compound and exhibits a remarkable bioprotective effect against desiccation and heat denaturation of functional protein complexes.

ESEEM measurements were performed at two temperatures on Photosystem I (PS I) complexes depleted of the extrinsic PsaC subunit harboring iron–sulfur 4Fe–4S clusters FA/FB (abbreviated FX-core complexes) embedded in trehalose glassy matrix. The ESEEM modulation frequencies obtained for the FX-core complexes in trehalose matrix were similar at 150 K and 290 K. They correspond to a distance ~ 25 A between the oxidized primary donor P700·+ and reduced phylloquinone acceptor A1·−. This distance corresponds to the preferential charge separation pathway between P700·+ and A1B·− in the B-branch of the PS I redox cofactors, while the distance estimated previously for the intact PS I complexes (containing the extrinsic PsaC subunit) embedded in dry trehalose matrix was ~ 26 A, which is typical for the formation of P700·+A1A·− charge-separated radical pairs in the symmetrical A-branch (Sukhanov et al. in Appl Magn Reson 49:1011, 2018). The redirection of electron transfer preferentially from the A to the B-branch of redox cofactors observed in FX-core complexes embedded in dry trehalose matrix can be rationalized by the alteration of the protein domain rigidity/flexibility around phylloquinone molecules in the A1A and A1B sites, which affects the thermodynamics of electron transfer between P700 and A1 in the symmetrical branches of cofactors. This finding of similar modulation frequencies measured for FX-core complexes in trehalose matrix at 150 K and 290 K is opposite to our previously obtained results for intact PS I, where the value of ESEEM modulation frequency markedly decreased upon heating the sample from cryogenic to room temperature (Sukhanov et al. in Appl Magn Reson 49: 1011, 2018). Our comparison of FX-core complexes with intact PS I complexes suggests that 4Fe-4S clusters accelerate spin–lattice relaxation of the unpaired electron spins on the phylloquinone in the A1-site. The result serves as additional argument in favor of the molecular model of the cryoprotective effect of trehalose matrix on the functioning of PS I by way of rigidity changes of the hydrogen-bonding network of the protein with the matrix that is based on the Le Chatelier–Braun principle. According to this thermodynamic principle, changes in both rigidity and flexibility are compensated to establish new thermodynamic equilibria with restored global balance of rigidity and flexibility that is typical within functioning protein complexes.

The one-sided Mobius band topology with its characteristic 180° twist has fascinated and inspired philosophers, artists and scientists since a long time. On the molecular level, only in the last 13 years a few chemistry groups succeeded to artificially create novel compounds with Mobius symmetry by theory-based molecular design and elaborate chemical synthesis. The interest in molecules with Mobius band symmetry was greatly stimulated in 1964 by a theoretical paper by Edgar Heilbronner from the ETH Zurich. He predicted that sufficiently large [n]annulenes with a closed-shell electron configuration of 4n π-electrons should allow for sufficient π-overlap stabilization to be synthesizable by twisting them into the Mobius topology of their hydrocarbon skeleton. In 2003, the first synthesis of an aromatic Mobius annulene was accomplished by Rainer Herges and co-workers in Kiel. In 2007, Lechoslaw Latos-Grazynski and co-workers in Wroclaw succeeded in synthesizing free-base di-p-benzi (Yoneda et al., Angew Chem Int Ed 53: 13169–13173, 2014) hexaphyrin (1.1.1.1.1.1), compound 1, an expanded porphyrin which can dynamically switch between Huckel and Mobius conjugation upon changes of solvent and temperature. Shortly thereafter, in 2008, Atsuhiro Osuka and his co-workers from Kyoto, Seoul and Hyogo published the synthesis of an expanded porphyrin in which metalation triggered the production of molecular twisting and Mobius aromaticity. In this minireview, among other studies also our recent EPR, ENDOR and DFT studies on open-shell states of 1, i.e., on the ground-state radical cation doublet state (total electron spin S = 1/2) and the first excited triplet state (S = 1) are summarized. The review is largely based on a previous joint publication of the current authors with the Latos-Grazynski group on radical cations of 1 (Mobius et al., Phys Chem Chem Phys 17:6644–6652, 2015). The radical cation study was the first one of an open-shell π-system with Mobius topology. In the doublet state, the hyperfine interactions of the unpaired electron spin with specific magnetic nuclei in the molecule was used as a sensitive probe for the electronic structure of the molecule and its symmetry properties. This work has now been extended to state-of-the-art DFT theory studies on photo-excited triplet states of 1. In the open-shell triplet state, besides hyperfine couplings, a change of the zero-field splitting interaction between the two unpaired electron spins is predicted to be a viable sensor for electronic structure changes upon Mobius-to-Huckel topology switching.

The structural and dynamical interaction of proteins with their microenvironment in disordered matrices plays a decisive role for their function; EPR spectroscopy is a powerful tool for shading light onto the molecular mechanisms of this protein–matrix interplay. To clarify the molecular mechanisms of disaccharide bioprotection, we studied the structure and dynamics of spin-labeled systems and photosynthetic reaction centers (RCs) in sucrose and trehalose matrices at different hydration levels by means of cw and pulse high-field 95 GHz (W-band) EPR as well as by FTIR. In this minireview, we summarize and discuss EPR and FTIR experiments showing that the anhydrobiotic state of the RC–trehalose system (1) is not the result of matrix-induced changes of the local structure of the charge-separated radical-pair cofactors, $${\text{P}}_{865}^{ \cdot + }$$ and $${\text{Q}}_{\text{A}}^{ \cdot - }$$ , and (2) is not the result of changes of local dynamics and local hydrogen bonding of QA in its binding pocket. Rather, the extreme impairment of RC dynamics caused by incorporation into the dehydrated trehalose matrix, which also protects it against thermal denaturation, originates in the high rigidity, already at room temperature, of the dry trehalose glass matrix coating the RC protein surface. This surface hydrogen-bonding scaffold shifts the correlation time of thermal conformational fluctuations into the non-biological time domain. Another intriguing aspect of disaccharide bioprotection is the superior efficiency of trehalose versus sucrose matrices in stabilizing the anhydrobiotic state of proteins. To clarify the molecular basis of this specificity, glassy trehalose–water and sucrose–water binary systems, incorporating a nitroxide radical as spin probe, have been studied by high-field W-band EPR spectroscopy at different water contents. Analysis of the EPR spectra revealed a different structural and dynamical organization in the sucrose and trehalose matrix, only the trehalose being homogeneous in terms of residual water and nitroxide distribution.

Photosystem I (PS I) has two nearly identical branches of electron-transfer co-factors. Based on point mutation studies, there is general agreement that both branches are active at ambient temperature but that the majority of electron-transfer events occur in the A-branch. At low temperature, reversible electron transfer between P(700) and A(1A) occurs in the A-branch. However, it has been postulated that irreversible electron transfer from P(700) through A(1B) to the terminal iron-sulfur clusters F(A) and F(B) occurs via the B-branch. Thus, to study the directionality of electron transfer at low temperature, electron transfer to the iron-sulfur clusters must be blocked. Because the geometries of the donor-acceptor radical pairs formed by electron transfer in the A- and B-branch differ, they have different spin-polarized EPR spectra and echo-modulation decay curves. Hence, time-resolved, multiple-frequency EPR spectroscopy, both in the direct-detection and pulse mode, can be used to probe the use of the two branches if electron transfer to the iron-sulfur clusters is blocked. Here, we use the PS I variant from the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, to incorporate 2,3-dichloro-1,4-naphthoquinone (Cl(2)NQ) into the A(1A) and A(1B) binding sites. The reduction midpoint potential of Cl(2)NQ is approximately 400 mV more positive than that of phylloquinone and is unable to transfer electrons to the iron-sulfur clusters. In contrast to previous studies, in which the iron-sulfur clusters were chemically reduced and/or point mutations were used to prevent electron transfer past the quinones, we find no evidence for radical-pair formation in the B-branch. The implications of this result for the directionality of electron transfer in PS I are discussed.

Photosystem I (PS I) mutants from the cyanobacterium Synechocystis sp. PCC 6803 bearing point mutations to the axial ligands of A0A (M688NPsaA) and A0B (M668NPsaB) were studied by high-field W-band electron paramagnetic resonance (EPR) spectroscopy. It was found that the EPR observables of PS I from the M668NPsaB mutant were virtual identical to that of the wild type (WT), and are clearly distinct from the M688NPsaA mutant. In particular, the P700·+ decay kinetics in the M688NPsaA mutant is significantly slower than in the WT or the M668NPsaB mutant. The analysis of the out-of-phase electron–electron dipolar electron spin echo envelope modulation shows that in the M668NPsaB mutant, the estimated distance of 26.0 ± 0.3 A agrees well with the 25.8 A distance for the P700·+A1A·− radical pair measured in the X-ray crystal structure. In the M688NPsaA mutant, two populations are found with estimated distances of 26.0 ± 0.3 and 25.0 ± 0.3 A in a ratio of 0.7–0.3, which agree well with the 25.8 A distance for the P700·+A1A·− radical pair and the 24.6 A distance for the P700·+A1B·− radical pair measured in the X-ray crystal structure. The data confirm that under the experimental conditions employed in this work, which involve dark-adapted samples without the pre-reduction of the iron–sulfur clusters, electron transport in cyanobacterial PS I is asymmetrical at 100 K, with the majority of electron transfer taking place through the A-branch of cofactors.

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g- and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g- and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90–240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.

In a recent paper (Bardelang et al. in J Org Chem 71:7657–7667, 2006), the deep inclusion of the TIPNO (1-phenyl-2-methylpropyl-1,1-dimethylethyl-2-nitroxide) free radical attached to a permethylated β-cyclodextrin (TRIMEB) into its cavity was questioned. In the present paper it is shown that the results of X-band and W-band electron paramagnetic resonance (EPR) and 1H nuclear magnetic resonance (NMR) studies of the TIPNO–TRIMEB in the presence of competitors of complexation (1-adamantanol, methyl orange and 1-adamantylamine) are consistent with a situation where the nitroxide is capping the small cavity entrance. The inclusion of the incoming guest was proven by 1H NMR, whereas no changes in EPR spectra were noticed whatever the competitors’ concentrations. These observations are rationalized in terms of an equilibrium between a nitroxide-capped cyclodextrin (weak complex) and a non-self included form, both species involving competitors’ inclusion without significant EPR spectral changes. These results not only confirm our preliminary findings, but also point out to the peculiar role of the cyclodextrin methoxy crown of the primary rim stabilizing the appended guest by means of weak interactions.

Among the numerous spectroscopic techniques utilized in photosynthesis research, high-field/high-frequency EPR and its pulse extensions ESE, ENDOR, ESEEM, and PELDOR play an important role in the endeavor to understand, on the basis of structure and dynamics data, dominant factors that control specificity and efficiency of light-induced electron- and proton-transfer processes in primary photosynthesis. Short-lived transient intermediates of the photocycle can be characterized by high-field EPR techniques, and detailed structural information can be obtained even from disordered sample preparations. The chapter describes how multifrequency high-field EPR methodology, in conjunction with mutation strategies for site-specific isotope or spin labeling and with the support of modern quantum-chemical computation methods for data interpretation, is capable of providing new insights into the photosynthetic transfer processes. The information obtained is complementary to that of protein crystallography, solid-state NMR and laser spectroscopy.

An improved TM110 resonator for continuous-wave and time-resolved electron-nuclear double resonance (ENDOR) studies at X-band frequencies is described that has been designed with small samples and/or light excitation in mind. The filling factor is increased by reducing the resonator length to only 16 mm. The radio-frequency field is generated by either a conventional solenoid for stable samples or by a pair of coils for samples which require photoexcitation. This arrangement leaves the central volume of the resonator free for optimal sample illumination, which is achieved through a slit in the wall of the resonator. Microwave coupling is achieved by the incorporation of an iris in the top of the resonator, and magnetic field modulation is applied by external coils. The resonator’s high sensitivity is illustrated by a study of the temperature dependence of the continuous-wave ENDOR signal of the stable neutral flavin radical in DNA photolyase and an investigation of the photoexcited triplet state of free-base tetraphenylbacteriochlorin by time-resolved ENDOR.

In this work, by using the respective advantages of W- and X-band electron paramagnetic resonance (EPR) spectroscopy techniques to investigate electron transport processes, we have studied the light-induced redox transients of the primary electron donor P700 and the secondary acceptor A1 in photosystem (PS) I complexes of intact cyanobacterial cellsSynechocystis sp. PCC 6803. We found that the kinetic behavior of the cation radical P700·+ generated by illumination with continuous light, and the EPR intensity of the radical pair P700·+A1·− generated upon laser pulse illumination strongly depend on the illumination prehistory (either the sample was frozen in the dark or during illumination). Both these processes were sensitive to the presence of electron transport inhibitors which block electron flow between the two photosystems. In line with our X-band EPR data on the kinetics of light-induced redox transients of P700, our high-field W-band EPR study of the radical-pair state P700·+A1·− shows that photosynthetic electron flow through the PS I reaction center is controlled both on the donor and on the acceptor side of PS I.

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT2 relaxation as well as by the dipolar interaction between P865•+QA•− radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P865•+QA•−, which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.

A computational model to simulate electron spin polarization in the three-spin-1/2 system composed of the molecular excited triplet state of (tetraphenylporphinato)zinc(II) (ZnTPP) and the doublet ground state of the 3-(N-nitronyl-notroxide) pyridine (3-NOPy) stable radical is proposed. The model is based on numerical solutions of the stochastic Liouville equation for the diffusively rotating system where the magnetic dipolar, isotropic Heisenberg exchange, and anisotropic Zeeman electron spin interactions are taken into account in a full measure, whereas the intersystem crossing processes between the singlet and triplet states of ZnTPP are considered in terms of kinetic equations for the relevant spin density matrices. Additional longitudinal and transversal paramagnetic relaxation caused by relative rotation motions of the ZnTPP and 3-NOPy moieties is taken into consideration in the form of the generalized relaxation operator.

Continuous-wave high-field electron paramagnetic resonance (95 GHz, 3.4 T) is performed on a spin label side chain located at residue position 171 in the proton entrance channel of bacterior-hodopsin The conformational differences of three bacteriorhodopsin mutants, the single mutant F171C, the double mutant D96G/F171C, and the triple mutant D96G/F171C/F219L, are reflected in different gxx and Azz tensor component shifts of the nitroxide side chain. The most polar microenvironment is found in the single mutant, whereas the open proton entrance channel reported for the triple mutant allows a reorientation of the nitroxide group towards a microenvironment of lower polarity and/or reduced hydrogen bonding. The experimental data of the double mutant are explained by a light-independent equilibrium of two nitroxide orientations with different polarities of the local microenvironment. Upon illumination the spectrum of the single mutant revealsg xx andA zz tensor component shifts which resemble those determined for the triple mutant in the dark. This result provides strong evidence for a light-induced opening of the proton entrance channel of the single mutant similar to that found in the unilluminated triple mutant, in agreement with electron diffraction data.

The photoexcited quartet (Q1) and doublet (D1) states of the complex of tetra-phenyl-porphine zinc (II) and a nitroxide radical have been studied in toluene solution by X-(9.4 GHz) and W-band (94 GHz) time-resolved electron paramagnetic resonance spectroscopy. The spin-polarized signals of the Q1 and D1 states are observed and assigned by analysis of theg-values. Line broadening and spin polarization mechanisms in this system are discussed.

We investigate the effect of stochastic modulation of the exchange interaction Jex on singlet (S)-triplet (T) transitions in radical pairs. These transitions limit the lifetime of the photo-generated radical pairs in covalently linked porphyrin-quinone systems that have been developed for biomimetic modeling of photosynthetic electron transfer processes. In order to explain transient electron paramagnetic resonance (EPR) results in different magnetic fields, i.e., with X-band ((0.34 T)/ (9.5 GHz)) and W-band ((3.4 T)/(95 GHz)) time-resolved EPR, we have to assume that Jex is modulated over a range of 20000 G, which is wide enough that S is temporarily almost degenerate with T0 as well as with T−1. This large modulation of Jex is caused by restricted rotational diffusion of the quinone subunit with respect to the porphyrin subunit. However, because of the small interradical distance of about 1.0–1.4 nm, the radical pair is continuously kept in the strong coupling limit and, therefore, we observe only EPR transition between the triplet sublevels. We find an approximation to solve the stochastic Liouville equation valid for rotational diffusion on an intermediate time scale, i.e., the diffusion rate DR is smaller than the singlet electron recombination rate KS ∼ 109 s−1, but larger than the ST transition rates κ0, κ−1 < 106 s−1.

The “Contracted Porphyrins” corrole and the recently synthesized isocorrole (as octaethyl derivatives) represent novel porphyrinoid macrocycles of biochemical interest. The radical cations of the free-bases of both corrole and its isomer are studied by EPR, ENDOR, and TRIPLE resonance in liquid solution to measure isotropic hyperfine couplings including signs. They yield detailed information of the electronic structure of the radical cations. The experimental findings are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/SP). The comparison shows that the free-bases of corrole and isocorrole undergo protonation when oxidized to cation radicals in CH2Cl2 solution. Excellent agreement of the measured and calculated spin density distributions is achieved for the protonated dication radicals, which exhibit the C2 symmetry observed for the hyperfine structure. The analysis of the hyperfine data discriminates against NH tautomerism as the sole source for an effective C2 symmetry of the macrocycle.

ENDOR spectra of triplet state molecules have a chacteristic line from degenerate NMR transitions within the zero level (ZL) electron spin manifoldM S=0. The ZL line, observed at the free nuclear Larmor frequency, dominates spectra when the number of nuclei is large. This line was found to be substantially reduced in intensity at low temperature. The strong variation of the ZL line intensity is analyzed within the frame of an electric-circuit analogy modeling. The result is as follows: At low temperature the electron and nuclear spin-lattice relaxation rates become small, and the nuclear-nuclear spin flip-flop transitions between degenerate substates,M I=const within the ZL manifold, become relatively strong to compete for population redistribution. This reduces the population differences between nuclear sublevels. Additional NMR irradiation can thus do very little to reduce these differences even more, and the ENDOR effect becomes suppressed. A certain enhancement of the relaxationally suppressed ZL line occurring at increased EPR saturation is explained by the coherent action of the microwave field on the selected substate within the ZL manifold that shifts its energy out of the other degenerate states thereby closing the flip-flop relaxation channel.

The specific ENDOR line at the free Larmor frequency νp in the low temperature spectra of triplet state molecules is caused by degenerate NMR transitions within theMs=0 zero-level (ZL) electron spin manifold. This ZL line was found to be orientationally dependent for the diradical complex Zn(3,6-di-tert-butyl-o-semiquinone)2Zn(DBSQ)2: the ZL line dominates the ENDOR spectrum if it is detected at the perpendicular canonical components of the EPR spectrum, and vanishes if the complex is oriented with its ZFSz-axis parallel to the direction of the magnetic field, i.e., if detected at the parallel canonical EPR components. This effect is shown to result from the interaction between nuclear spin substates of the S and T0 manifolds, their levels being close to each other for the Zn(DBSQ)2 complex. Such an interaction mixes the states and shifts energy levels. Consequently, it cancels the degeneracy of the nuclear substates within the ZL manifold and reduces the rate of nuclear flip-flop relaxation. This specific relaxation mechanism has been shown to substantially affect the amplitude of the ZL line (Doubinskii A.A., Lebedev Ya.S., Mobius K.: Appl. Magn. Reson.13, 439 (1997)). The nuclear flip-flop relaxation effect is expected to be orientationally dependent since the S-To separation depends upon the orientation of the diradical with respect to the external magnetic field.

The electronic structure of the primary electron donor (D) of the photosynthetic bacteriumRhodobacter sphaeroides is investigated by ENDOR in the photoinduced triplet state of D. Hyperfine (hf) splittings of the triplet state measured on frozen solutions are given and compared to the results obtained earlier (Lendzian F., van Willigen H., Sastry S., Mobius K., Scheer H., Feick R.: Chem. Phys. Lett.118, 145 (1985). The hf splittings found are consistent with a model of an asymmetric spin density distribution over the two bacteriochlorophyll molecules which constitute D, suggesting a mirror image symmetry of HOMO and LUMO coefficients. The could be relevant for electron transfer, in particular unidirectionality in the reaction center (RC). The first triplet state ENDOR experiments on single crystals of RC’s are also reported.

An EPR and ENDOR probehead with a loop-gap resonator for X-band is described. The novel feature of the construction is that an iris-type coupling of the resonator is used instead of the conventional antenna coupling. The ENDOR coil combines the role of creating the radio frequency field and that of a shield for the microwave loop-gap structure. Hence, in order to accommodate the iris and waveguide, a pair of RF coils is used in conjunction with a reduced waveguide with dielectric filling. This arrangement simplifies matching the resonator to the microwave bridge, and standard EPR cryostats can be used making sample manipulation more convenient.

The bacteriochlorophyll (BGhl)a cation radical is studied by cw-EPR and multinuclear ENDOR/TRIPLE resonance techniques in liquid solution yielding the hyperfine coupling constants (hfcs) for 12 protons, all 4 nitrogens and the central magnesium nucleus. The H hfcs are assigned to specific molecular positions by various experimental techniques including isotope substitution, H/D exchange experiments and comparison with bacteriochlorophyll derivatives. The determined temperature dependences of the H hfcs are measured and related to dynamical properties of the molecule in solution. The experimental hfcs compare well with those obtained from semiempirical molecular orbital calculations of the RHF-INDO/SP type. The importance of these results for BChl speciesin vivo, i.e., in the protein matrix, is discussed.

Fourier transform EPR spectroscopy was employed in studying the electron transfer (ET) reaction and the quenching mechanisms of the photoexcited triplet state of C60 as electron acceptor and N,N,N′,N′-tetramethylbenzidine (NTMB) as electron donor in benzonitrile solution. The ET reaction product, the cation radical NTMB*+, interacts with 3*C60, leading to photoinduced electron polarization of NTMB*+ via triplet-doublet mixing mechanism combined with triplet mechanism. The quenching of 3*C60 and the polarization behavior of NTMB*+ are discussed.

Radicals generated photochemically from a covalently linked porphyrin-quinone donor-acceptor system dissolved in reversed micelles and isotropic solution have been studied by steady-state and time-resolved EPR spectroscopy. In these systems photoinducedintramolecular as well asintermolecular electron transfer processes occur which result in the formation of semiquinone radical anions and porphyrin radical cations. Disproportionation of the semiquinone leads to the formation of porphyrin-hydroquinone (and porphyrin-quinone). The porphyrin-hydroquinone is itself photoactive and reacts through the photoexcited triplet state of the porphyrin. Reduction of the porphyrin to the dihydro from — probablyvia hydrogen abstraction by the photoexcited porphyrin from the hydroquinone — appears to be the dominant reaction. Once formed the dihydrophyrin undergoes further similar photochemistry. Emissively polarized spectra are observed from these systems in steady-state EPR experiments. Timeresolved EPR indicates that this polarization is essentially due to the radical triplet pair mechanism.

In photosynthesis research the elucidation of the spatial and electronic structures of the electron donor and acceptor ion radicals is very important for an understanding of the light-induced electron transfer process. Recent 3 mm (W-band, 95 GHz) high-field EPR and ENDOR studies on the primary donor cation radicals P+. (bacteriochlorophyll dimer), the acceptor anion radicals Q+. (quinones), and the charge-separated radical pair (P+.Q−.) in photosynthetic bacteria and biomimetic model systems are presented. From single crystals of, for instance,Rb. sphaeroides R-26 reaction centers both the hyperfine tensors of various protons and theg-tensor of P865 +. have been determined and compared with calculated tensor values based on recent X-ray structure data. The results consistently reveal a breaking of the local C2 symmetry of the electronic structure at the primary donor side of the reaction center. This is of particular interest since it might be relevant for the vectorial electron transfer along the protein complex. Among the quinone radical anions studied are frozen solutions of the electron acceptors of bacterial and plant reaction centers (ubiquinone and plastoquinone, respectively). The increased electron Zeeman interaction in high-field EPR leads to almost completely resolvedg-tensor components even in disordered samples. Theg-tensors and component linewidths are sensitive probes for specific anisotropic interactions with the environment. In the case of the transient correlated coupled radical pair P865 +.-QA −. ofRb. sphaeroides (Fe replaced by Zn) the spin-polarized high-field EPR spectra allow an unambiguous determination of the relative orientation of theg-tensors of the donor and acceptor parts. Thereby high-precision structure information is obtained on the electron transfer pigments after light-induced charge separation.

The origin of frequently observed “negative” (opposite phase) ENDOR lines in the low-frequency region of triplet state ENDOR spectra is explained in terms of microwave hole burning and RF modulation phenomena. From this, a new method of detecting burnt side holes in EPR spectra is derived which is based on cw ENDOR instrumentation. The method uses the modulation satellites that are induced by a longitudinal RF field component and appear around any EPR line, including burnt holes (“negative” lines). The longitudinal RF field was generated by a coil oriented parallel to the external field, but a longitudinal component of the RF field also exists in most conventional ENDOR spectrometers because of slight misalignments of the ENDOR coil generating the transversal RF field. The lines it induces in the low-frequency part of ENDOR spectra are generally considered as artifacts. It is shown, however, that RF induced modulation satellites provide valuable information concerning the lines distant from the spectral position in the EPR spectrum chosen for ENDOR observation. This allows one to record the pattern of side holes burnt by microwave saturation through forbidden transitions that carries information about ENDOR frequencies comparable to what can be extracted from ESEEM experiments. Such comparability is demonstrated for examples of nitrogen ENDOR of photoexcited triplet states of the primary donor in photosynthetic reaction centers and related compounds.

Various time resolved EPR methods are applied to different test samples to demonstrate the abilities of pulsed high-field EPR spectroscopy. Two-pulse-echo field swept EPR spectroscopy on a nitroxide radical shows the increased spectral resolution by separating different spin systems by their relaxation properties. Additionally N14 electron-spin-echo-envelope-modulation (ESEEM) is observed for these systems at fields as high as 3.5 T. Thus, the N14 hyperfine interaction couplings can be probed by ESEEM and pulsed ENDOR (electron-nuclear-double-resonance) experiments. The sensitivity of pulsed ENDOR experiments is compared with cw-ENDOR. The different linewidths and amplitudes of the two methods are discussed. Transient nutation experiments on light induced triplet states demonstrate the high sensitivity and time resolution of high-field pulsed EPR. The sensitivity and time resolution of our 95 GHz spectrometer are determined and compared with pulsed X-band EPR spectrometer performances.

We report on time-resolved EPR experiments of the photo-induced electron transfer from zinc-tetraphenylporphyrin (ZnTPP) to duroquinone (DQ) in cationic CTAC and neutral Triton X-100 micelles. The spin-polarized EPR spectra and their time-dependence indicate pronounced differences between the two micellar systems: In the neutral micelles, the lifetime of the spin-correlated radical pair is longer than in the charged micelles. In the CTAC system an unusual temperature dependence of the polarization pattern is observed. This can be attributed to the effects of both the microviscosity of the micellar interior and the macroviscosity of the bulk solution on the spin dynamics of the reactants located inside the micelles.

The triplet states of several substituted porphyrins (Tetraphenylporphyrin (H2TPP), Zinc-Tetramethylporphyrin (ZnTMP), Octaethylporphyrin (H2OEP) and the Dication of H2TPP (H4TPP2+)) and two covalently linked dimers with H2TPP-subunits in disordered solid solution were studied by EPR and ENDOR at liquid helium temperature. The measurement yields theAzz component of the hyperfine tensors of all α-protons in the reference frame of the zero field splitting tensor. Dipolar and isotropic contributions toAzz are discussed and spin densities derived. The spin densities are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/S). One of the dimers shows indications of triplet energy transfer between the porphyrin subunits. The order of magnitude of the transfer rate is estimated to be 5 · 105 s−1.

Porphycene is a structural isomer of porphyrin. The photoexcited triplet states of porphycene, 2,7,12,17-tetra-n-propylporphycene and 9,10,19,20-tetra-n-propylporphycene in disordered solid solution were studied by EPR and ENDOR. The ENDOR spectra yield the hyperfine tensor elementsA zz for each of the different groups of equivalent protons. The dipolar contribution toA zz is estimated and spin densities are derived from the isotropic contribution. They are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/SP and RHF-INDO/S).

The oxidized primary electron donor P865+ in reaction center single crystals of Rb. sphaeroides wild type 2.4.1 has been investigated by electron-nuclear-nuclear TRIPLE resonance techniques. The measured anisotropic and isotropic hyperfine couplings have been compared with data from the carotenoidless mutant R-26 of the same organism. There is no detectable structural difference of the dimer P865+ in both strains and its orientation in the crystal unit cell axes is the same within ±3°. However, a small shift of spin density towards the L-half of the dimer is observed due to the presence of the carotenoid.

From time-resolved direct detection cw EPR with pulsed laser excitation, the photoinduced electron transfer and spin dynamics (CIDEP) in mixed zinc-tetraphenylporphyrin (ZnTPP)/benzo-1,4-quinone (BQ) ethanol solutions were determined as functions of temperature and BQ concentration. At lower temperatures the EPR spectra reveal that mixing of the S and T−1 states in the charge separated radical pair gains in importance relative to the ST0 mixing. Furthermore, at lower temperatures, the EPR spectra of the spin-correlated radical pairs of ZnTPP+ and BQ7 could also be observed. From the temperature/viscosity dependence of the electron transfer rates and of the polarization contributions from the triplet and radical pair mechanisms, deviations from a macroscopic diffusion behaviour are inferred at lower temperatures.